Publications and other materials including patents and patent applications used to illuminate the specification are hereby incorporated by reference.
Amylin
Amylin is a 37 amino acid protein hormone. It was isolated, purified and chemically characterized as the major component of amyloid deposits in the islets of pancreases of human Type II diabetics (Cooper et al., Proc. Natl. Acad. Sci., USA 84:8628-8632 (1987)). The amylin molecule has two important post-translational modifications: the C-terminus is amidated, and the cysteines in positions 2 and 7 are cross-linked to form an N-terminal loop. Amylin is the subject of United Kingdom patent application Serial No. 8709871, filed Apr. 27, 1987, and corresponding U.S. Pat. No. 5,367,052, issued Nov. 22, 1994.
Amylin is a member of a family of related peptides which include CGRP and calcitonin (Rink et al., Trends Pharmacol. Sci. 14:113-118 (1993)). Amylin is primarily synthesized in pancreatic beta cells and is secreted in response to nutrient stimuli such as glucose and arginine. Moore et al., Biochem. Biophys. Res. Commun. 179:1-9 (1991); Kanatsuka et al., FEBS Lett. 259:199-201 (1989); Ogawa et al., J. Clin. Invest. 85:973-976 (1990); Gedulin et al., Biochem. Biophys. Res. Commun. 180:782-789 (1991).
In normal humans, fasting amylin levels from 1 to 10 pM and post-prandial levels of 5 to 20 pM have been reported (e.g., Hartter et al., Diabetologia 34:52-54 (1991); Sanke et al., Diabetologia 34:129-132 (1991)); Koda et al., The Lancet 339:1179-1180 (1992)). In obese, insulin-resistant individuals, however, post-food amylin levels can go higher, reaching up to about 50 pM, for example. For comparison, the values for fasting and post-prandial insulin are 20 to 50 pM, and 100 to 300 pM respectively in healthy people, with perhaps 3-to 4-fold higher levels in insulin-resistant people. In Type 1 diabetes, where beta-cells are destroyed, amylin levels are at or below the levels of detection and do not rise in response to glucose (Koda et al., The Lancet 339:1179-1180 (1992)). In normal mice and rats, basal amylin levels have been reported from 30 to 100 pM, while values up to 600 pM have been measured in certain insulin-resistant, diabetic strains of rodents (e.g., Huang et al., Hypertension 19:I-101-I-109 (1991)); Gill et al., Life Sciences 48:703-710 (1991).
The first discovered action of amylin was the reduction of insulin-stimulated incorporation of glucose into glycogen in rat skeletal muscle (Leighton et al., Nature 335:632-635 (1988)); the muscle was made "insulin-resistant". Subsequent work with rat soleus muscle ex-vivo and in vitro has indicated that amylin reduces glycogen-synthase activity, promotes conversion of glycogen phosphorylase from the inactive b form to the active a form, promotes net loss of glycogen (in the presence or absence of insulin), increases glucose-6-phosphate levels, and can increase lactate output (see, e.g., Deems et al., Biochem. Biophys. Res. Commun. 181:116-120 (1991)); Young et al., FEBS Letts. 281:149-151 (1991)).
It is believed that amylin acts through receptors present in plasma membranes. Beaumont et al., Mol. Pharmacol. 44:493-497 (1993). Amylin receptors and their use in various methods for screening and assaying for amylin agonist and antagonist compounds are described in U.S. Pat. No. 5,264,372, issued Nov. 23, 1993.
The biological actions of amylin relating to fuel metabolism are discussed in Young et al., J. Cell. Biochem. 555:12-18 (1994). While amylin has marked effects on hepatic fuel metabolism in vivo, there is no general agreement as to what amylin actions are seen in isolated hepatocytes or perfused liver. The available data do not support the idea that amylin promotes hepatic glycogenolysis, i.e., it does not act like glucagon (e.g., Stephens, et al., Diabetes 40:395-400 (1991)); Gomez-Foix et al., Biochem J. 276:607-610 (1991)). It has been suggested that amylin may act on the liver to promote conversion of lactate to glycogen and to enhance the amount of glucose able to be liberated by glucagon (see Roden et al., Diabetologia 35:116-120 (1992)). Thus, amylin could act there as an anabolic partner to insulin in liver, in contrast to its catabolic action in muscle.
In fat cells, contrary to its action in muscle, amylin has no detectable actions on insulin-stimulated glucose uptake, incorporation of glucose into triglyceride, CO.sub.2 production (Cooper et al., Proc. Natl. Acad. Sci. 85:7763-7766 (1988)) epinephrine-stimulated lipolysis, or insulin-inhibition of lipolysis (Lupien and Young, Diabetes Nutrition and Metabolism--Clinical and Experimental, Vol. 6(1), pages 13-18 (1993)). Amylin thus exerts tissue-specific effects, with direct action on skeletal muscle, marked indirect (via supply of substrate) and perhaps direct effects on liver, while adipocytes appear "blind" to the presence or absence of amylin. No direct effects of amylin on kidney tissue have been reported.
It has also been reported that amylin can have marked effects on secretion of insulin. In isolated islets (Ohsawa et al., Biochem. Biophys. Res. Commun. 160:961-967 (1989)), in the perfused pancreas (Silvestre et al., Reg. Pept. 31:23-31 (1991)), and in the intact rat (Young et al., Mol. Cell. Endocrinol. 84:R1-R5 (1992)), various experiments indicate that amylin down-regulates insulin secretion. The perfused pancreas experiments point to selective down-regulation of the secretary response to glucose with sparing of the response to arginine. Other workers, however, have been unable to detect effects of amylin on isolated beta cells, on isolated islets, or in the whole animal (see Broderick et al., Biochem. Biophys. Res. Commun. 177:932-938 (1991) and references therein).
A striking effect of amylin in rodents at pharmacological dosages in vivo is stimulation of a sharp rise in plasma lactate, followed by a rise in plasma glucose (Young et al., FEBS Letts. 281:149-151 (1991)). Evidence indicates that the increased lactate provides substrate for glucose production and that amylin actions can occur independent of changes in insulin or glucagon. In "glucose clamp" experiments, amylin infusions cause "insulin resistance", both by reducing peripheral glucose disposal, and by limiting insulin-mediated suppression of hepatic glucose output (e.g., Frontoni et al., Diabetes 40:568-573 (1991)); Koopmans et al., Diabetologia 34:218-224 (1991)).
It has been shown that amylin agonists can slow gastric emptying (Young et al., Diabetologia (June 1995, in press), and is believed to contribute to their ability to reduce post-prandial hyperglycemia (Moyses and Kolterman, Drugs of the Future (May 1995). Methods for reducing gastric motility and slowing gastric emptying comprising the administration of an amylin agonist (including amylin) are the subject of United States patent application Ser. No. 08/118,381, filed Sep. 7, 1993, and U.S. patent application Ser. No. 08/302,069, filed Sep. 7, 1994 (and corresponding PCT application, Publication No. WO 95/07098, published Mar. 16, 1995).
Non-metabolic actions of amylin include vasodilator effects which may be mediated by interaction with CGRP vascular receptors. (Brain et al., Eur. J. Pharmacol. 183:2221 (1990). It has also been reported that amylin markedly increases plasma renin activity in intact rats when given subcutaneously in a manner that avoids any disturbance of blood pressure. Methods for treating renin-related disorders with amylin antagonists are described in U.S. Pat. No. 5,376,638, issued Dec. 27, 1994.
Injected into the brain, amylin has been reported to suppress food intake (e.g., Chance et al., Brain Res. 539:352-354 (1991)), an action shared with CGRP and calcitonin. The effective concentrations at the cells that mediate this action are not known. Amylin has also been reported to have effects both on isolated osteoclasts where it caused cell quiescence, and in vivo where it was reported to lower plasma calcium by up to 20% in rats, in rabbits, and in humans with Paget's disease (see, e.g., Zaidi et al., J. Bone Mineral Res. 5(Suppl. 2) 576 (1990). From the available data, amylin seems to be 10 to 30 times less potent than human calcitonin for these actions. Interestingly, it was reported that amylin appeared to increase osteoclast cAMP production but not to increase cytosolic Ca.sup.2+, while calcitonin does both (Alam et al., Biochem. Biophys. Res. Commun. 179:134-139 (1991)). It was suggested, though not established, that calcitonin may act via two receptor types and that amylin may interact with only one of these.
Diabetes Mellitus
Diabetes mellitus is a serious metabolic disease that is defined by the presence of chronically elevated levels of blood glucose. Classic symptoms of diabetes mellitus in adults are polyuria, polydipsia, ketonuria, rapid weight loss together with elevated levels of plasma glucose. Normal fasting plasma glucose concentrations are less than 115 milligrams per deciliter. In diabetic patients, fasting concentrations are found to be over 140 milligrams per deciliter. In general, diabetes mellitus develops in response to damage to the beta cells of the pancreas. This damage can result from primary diabetes mellitus, in which the beta cells are destroyed by the autoimmune system, or as a secondary diabetic response to other primary diseases, such as pancreatic disease, hormonal abnormalities other than lack of insulin action, drug or chemical induction, insulin receptor abnormalities, genetic syndromes or others. Primary diabetes mellitus can be classified as Type I diabetes (also called insulin-dependent diabetes mellitus or IDDM) and Type II diabetes mellitus (also called non-insulin dependent diabetes mellitus or NIDDM).
Type I (juvenile onset or insulin-dependent) diabetes is a well-known hormone deficient state, in which the pancreatic beta cells appear to have been destroyed by the body's own immune defense mechanisms. Patients with Type I diabetes mellitus have little or no endogenous insulin secretory capacity. These patients develop extreme hyperglycemia. Type I diabetes was fatal until the introduction of insulin replacement therapy some 70 years ago--first using insulins from animal sources, and more recently, using human insulin made by recombinant DNA technology. As discussed above, it is now clear that the destruction of beta cells in Type I diabetes leads to a combined deficiency of two hormones, insulin and amylin. When pancreatic cells are destroyed, the capacity to secrete insulin and amylin is lost. Type I diabetic subjects have reported amylin levels that are either undetectable or at the lower limit of detection, and which fail to increase in response to a glucose challenge. Koda, The Lancet 339:1179 (1992).
The nature of the lesion of the pancreatic beta cells in Type II diabetes is not clear. Unlike the pancreatic beta cells in Type I diabetics, the beta cells of Type II diabetics retain the ability to synthesize and secrete insulin and amylin.
Type II diabetes is characterized by insulin and resistance, i.e., a failure of the normal metabolic response of peripheral tissues to the action of insulin. In other words, insulin resistance is a condition where the circulating insulin produces a subnormal biological response. In clinical terms, insulin resistance is present when normal or elevated blood glucose levels persist in the face of normal or elevated levels of insulin. The hyperglycemia associated with Type II diabetes can sometimes be reversed or ameliorated by diet or weight loss sufficient to restore the sensitivity of the peripheral tissues to insulin. Indeed, Type II diabetes mellitus is often characterized by hyperglycemia in the presence of higher than normal levels of plasma insulin. Progression of Type II diabetes mellitus is associated with increasing concentrations of blood glucose and coupled with a relative decrease in the rate of glucose-induced insulin secretion. Thus, for example, in late- stage Type II diabetes mellitus, there may be an insulin deficiency.
The primary aim of treatment in all forms of diabetes mellitus is the same, namely the reduction of blood glucose concentrations to as near normal as possible, thereby minimizing both the short- and long-term complications of the disease. Tchobroutsky, Diabetologia 15:143-152 (1978). The linkage between the extent of hyperglycemia in diabetics and the ensuing long-term complications was further confirmed in the recently completed Diabetes Control and Complications Trial (DCCT) undertaken by the National Institutes of Health. The Diabetes Control and Complications Trial Research Group, N. Eng. J. Med. 329:977 (1993). The DCCT was conducted over a 10-year period at 29 clinical centers around the United States and Canada, and showed that lowering mean blood glucose concentrations in Type I diabetics reduced end-organ complications. The development of retinopathy was reduced by 76%, the progression of retinopathy by 54%, and there was an amelioration of the markers of renal disease (proteinuria, albuminuria). The development of significant neuropathic changes was also reduced.
The treatment of Type I diabetes necessarily involves the administration of replacement doses of insulin, administered by the parenteral route. In combination with the correct diet and self-blood glucose monitoring, the majority of Type I diabetics can achieve a certain level of control of blood glucose.
In contrast to Type I diabetes, treatment of Type II diabetes frequently does not require the use of insulin. Institution of therapy in Type II diabetes usually involves a trial of dietary therapy and lifestyle modification, typically for 6-12 weeks in the first instance. Features of a diabetic diet include an adequate but not excessive total calorie intake, with regular meals, restriction of the content of saturated fat, a concomitant increase in the polyunsaturated fatty acid content, and an increased intake of dietary fiber. Lifestyle modifications include the maintenance of regular exercise, as an aid both to weight control and also to reduce the degree of insulin resistance. If after an adequate trial of diet and lifestyle modifications, fasting hyperglycemia persists, then a diagnosis of "primary diet failure" may be made, and either a trial of oral hypoglycemic therapy or direct institution of insulin therapy will be required to produce blood glucose control and, thereby, to minimize the complications of the disease. Type II diabetics who fail to respond to diet and weight loss may respond to therapy with oral hypoglycemic agents such as sulfonylureas or biguanides. Insulin therapy, however, is used to treat other patients with Type II diabetes, especially those who have undergone primary dietary failure and are not obese, or those who have undergone both primary diet failure and secondary oral hypoglycemic failure.
The use of amylin agonists in the treatment of diabetes mellitus has been described in U.S. Pat. Nos. 5,124,314 and 5,175,145. Excess amylin action mimics key features of Type II diabetes and amylin blockade has been proposed as a novel therapeutic strategy. U.S. Pat. No. 5,266,561, issued Nov. 30, 1993, and U.S. Pat. No. 5,281,581, issued Jan. 25, 1994, disclose the treatment of Type II diabetes and insulin resistance with amylin antagonists.
It has previously been reported that infusion of the amylin agonist, .sup.25,28,29 Pro-h-amylin, decreased post-prandial glucose concentrations in Type I diabetics. Moyses & Kolterman, Drugs of the Future (May 1995). In a single blind crossover study in 6 male subjects with Type I diabetes, a 2-hour infusion of the amylin agonist, .sup.25,28,29 Pro-h-amylin at a rate of 150 .mu.g/hr was reported to significantly decrease post-prandial hyperglycemia following ingestion of a mixed meal. This effect was confirmed in a second study which employed an infusion rate of 50 .mu.g/hr for 5 hours in 9 subjects with Type I diabetes. A nutrient challenge was given either orally (in the form of a Sustacal.RTM. meal) or as an intravenous glucose load of 300 mg/kg. Administration of .sup.25,28,29 Pro-h-amylin reportedly led to a significant decrease in post-prandial glucose excursion after the oral Sustacal.RTM. meal, but not after the intravenous administration of glucose, consistent with an effect to delay gastric emptying and thus the gastrointestinal absorption of nutrients. In a further double-blind, placebo-controlled study, self-injected .sup.25,28,29 Pro-h-amylin, taken before meals thrice daily, also led to reduced glucose excursion after a test meal. The patients continued their usual insulin regime. In the third study, a statistically significant effect was seen with a dose of 30 .mu.g of the amylin agonist.